Ubiquitin Mediated Protein Degradation
Ubiquitin is known to be one of several factors required for ATP-dependent protein degradation in eukaryotic cells. One function of intracellular protein degradation, most of which is ATP-dependent, is selective elimination of damaged and otherwise abnormal proteins. Another is to confer short half-lives on undamaged proteins whose concentrations in the cell must vary as functions of time, as is the case, for example, with many regulatory proteins. Many other proteins, while long-lived as components of larger macromolecular complexes such as ribosomes and oligomeric proteins, are metabolically unstable in a free, unassociated state. Ubiquitination is also involved in the control of cell surface receptors such as platelet-derived growth factor (PDGF), the T cell receptor, G protein-coupled receptors and others. In addition to these proteins complexed with ubiquitin, ubiquitin is also found covalently linked to lipids in membranes (Guarino, L A, 1995, Cell 80, 301-309).
Ubiquitin, a 76-residue protein, is present in eukaryotes either free or covalently joined, through its carboxyl-terminal glycine residue, to various cytoplasmic, nuclear, and integral membrane proteins. A family of ubiquitin-conjugating enzymes (also called E2 enzymes) catalyzes the coupling of ubiquitin to such proteins (ubiquitination) generally in combination with a recognition element called E3 that may also function to carry out the ubiquitination. The fact that the protein of ubiquitin is conserved among eukaryotes to an extent unparalleled among known proteins suggests that ubiquitin mediates a basic cellular function.
It has been shown that selective degradation of many short-lived proteins requires a preliminary step of ubiquitin conjugation to a targeted proteolytic substrate. One role of ubiquitin is to serve as a signal for attack by proteases specific for ubiquitin-protein conjugates (Finley and Varshavsky, Trends Biochem. Sci. 10:343-348 (1985)).
At least some short-lived proteins are recognized as such because they contain sequences (degradation signals) which make these proteins substrates of specific proteolytic pathways. The first degradation signal to be understood in some detail comprises two distinct determinants: the protein's amino-terminal residue and a specific internal lysine residue, the N-end rule (Bachmair et al., Science 234:179-186 (1986); Bachmair and Varshavsky, Cell 56:1013-1032 (1989)). The N-end rule, a code that relates the protein's metabolic stability to the identity of its amino-terminal residue (Bachmair et al., Science 234:179-186 (1986), is universal in that different versions of the N-end rule operate in all of the eukaryotic organisms examined, from yeast to mammals (Gonda et al., J. Biol. Chem. 264:16700-16712 (1989)).
The second essential determinant of the N-end rule-based degradation signal, referred to as the second determinant, is a specific internal lysine residue in the substrate protein that serves as the site of attachment of a multiubiquitin chain. Formation of the multiubiquitin chain on a targeted short-lived protein is essential for the protein's subsequent degradation. The enzymatic conjugation of ubiquitin to other proteins involves formation of an isopeptide bond between the carboxy-terminal glycine residue of ubiquitin and the epsilon-amino group of a lysine residue in an acceptor protein. In a multiubiquitin chain, ubiquitin itself serves as an acceptor, with several ubiquitin moieties attached sequentially to an initial acceptor protein to form a chain of branched ubiquitin-ubiquitin conjugates (Chau et al., Science 243:1576-1583 (1989)).
The elucidation of the fundamental rules governing the metabolic stability of proteins in cells, and especially the deciphering of the N-end rule-based degradation signal, has made possible the manipulation of proteins to vary their half-lives in vivo (Bachmair and Varshavsky, Cell 56:1019-1032 (1989)).
The N-degron is an intracellular degradation signal whose essential determinant is a specific ("destabilizing") N-terminal amino acid residue of a substrate protein. A set of N-degrons containing different destabilizing residues is manifested as the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. The fundamental principles of the N-end rule, and the proteolytic pathway that implements it, are well established in the literature (see, e.g., Bachmair et al., Science 234: 179 (1986); Varshavsky, Cell 69: 725 (1992), U.S. Pat. Nos.: 5,122,463; 5,132,213; 5,093,242 and 5,196,321) the disclosures of which are incorporated herein by reference in their entirety.
In eukaryotes, the N-degron comprises at least two determinants: a destabilizing N-terminal residue and a specific internal lysine residue (or residues). The latter is the site of attachment of a multiubiquitin chain, whose formation is required for the degradation of at least some N-end rule substrates. Ubiquitin is a protein whose covalent conjugation to other proteins plays a role in a number of cellular processes, primarily through routes that involve protein degradation.
In a stochastic view of the N-degron , each internal lysine of a protein bearing a destabilizing N-terminal residue can be assigned a probability of being utilized as a multiubiquitination site, depending on time-averaged spatial location, orientation and mobility of the lysine. For some, and often for all of the Lys residues in a potential N-end rule substrate, this probability is infinitesimal because of the lysine's lack of mobility and/or its distance from a destabilizing N-terminal residue.
It is possible to construct a thermolabile protein bearing a destabilizing N-terminal residue in such a way that the protein becomes a substrate of the N-end rule pathway only at a temperature high enough to result in at least partial unfolding of the protein. This unfolding activates a previously cryptic N-degron in the protein by increasing exposure of its (destabilizing) N-terminal residue, by increasing mobilities of its internal Lys residues, or because of both effects at once. Since proteolysis by the N-end rule pathway is highly processive, any protein of interest can be made short-lived at a high (nonpermissive) but not at a low (permissive) temperature by expressing it as a fusion to the thus engineered thermolabile protein, with the latter serving as a portable, heat-inducible N-degron module.
The heat-inducible N-degron module can be any protein or peptide bearing a destabilizing N-terminal residue that becomes a substrate of the N-end rule pathway only at a temperature high enough to be useful as a nonpermissive temperature.
The idea of metabolically destabilizing a protein or peptide of interest using a protein or peptide (ie targeting a protein or peptide for degradation) has been described in U.S. Pat. No. 5,122,463. This metabolic destabilization requires that the protein or peptide of interest must contain a second determinant of the N-end rule-based degradation signal. The method comprises contacting the protein or peptide of interest with a targeting protein or peptide that interacts specifically with the protein or peptide of interest. The targeting peptide or protein is characterized as having a destabilizing amino-terminal amino acid according to the N-end rule of protein degradation.
The ability to activate the ubiquitination and degradation of other proteins not containing an N-terminus N-degron signal has been shown in a multisubunit protein where the N-degron signals are located on different subunits and still target a protein for destruction (U.S. Pat. No. 5,122,463). Moreover, in this case (trans recognition) only the subunit that bears the second N-degron signal (lysine) determinant is actually degraded. Thus, an oligomeric protein can contain both short-lived and long-lived subunits. In these examples the demonstrations are all based on known multisubunit proteins and alterations of these to bring about the destabilization of subunits involved in these multisubunit complexes.
A different aspect of targeting the ubiquitination system based on chimeric proteins of E2 to achieve selective targeting and alterations in the levels of proteins has been described (Gosink M M and Vierstra R D, 1995, Proc. Natl. Acad. Sci. 92, 9117-9121). These researchers demonstrated that selective ubiquitination and degradation can be achieved using a protein, which is a fusion protein of a ubiquitinating protein with a binding protein.
In one interesting study of the N end rule, the degradation of DHFR was stabilized by the binding of a small molecule indicating that binding small molecules could prevent the degradation of proteins. This was also suggested in U.S. Pat. No. 5,122,463 where the idea of using peptides and proteins to target the ubiquitination of proteins to which they bind is suggested. In this patent the peptides are described as binding in such a way that the peptide interferes with the folding of the target protein "folding-interfering targeting peptides" suggesting also that peptides binding might prevent degradation as seen with DHFR. Indeed in this patent the focus for the peptides is the sequence of the target protein to give rise to these destabilizing residues.
Other Protein Covalent Modification for Protein Targeting
A number of systems mirror the protein modification pathway of ubiquitin. Among these are based on the attachment of Apg12, Rub1/Nedd8 and Smt3/SUMO-1 to proteins in addition to the ubiquitin pathway. In these systems homology at the level of sequence is seen but also clear parallels can be drawn based on the functional elements involved in the various systems (S Jentsch and H. D. Ulrich, Nature (1998) 395, 321-322).
In the case of the Apg12 system this protein is involved in the autophagy of various cellular components. Apg12 appears to be the functional homologue of ubiquitin and is transferred via Apg7 and Apg10 the functional homologue of the E1 and E2 ubiquitin conjugating enzymes, respectively. Apg12 transferred via Apg7 and Apg10 is used to modify Apg5 to activate autophagy. The analysis of the sequence of Apg7 shows a considerable homology to the E1 enzymes of the ubiquitin pathway. In the case of Rub1/Nedd8 system this protein is involved in some regulatory role. The Smt3/SUMO-1 system is involved in the targeting of proteins.
Drug Targets
The number of drug targets for human therapeutics is around 400 human gene products, such as enzymes, receptors and ion channels. But there may be 2500-5000 molecular targets whose exploitation may be capable of restoring function in the 100 or so common human polygenic diseases. Many of these new targets are being discovered by the intensive search of the human genome by various groups using focused and random methods.
The following are examples of drug targets which are the subject of investigation by various pharmaceutical companies: B7.1 and B7, TNFR1m(p55), TNFR2 (p75), NADPH oxidase, Bc1/Bax and other partners in the apotosis pathway, C5a receptor, HMG-CoA reductase, PDE V phosphodiesterase type, PDE IV phosphodiesterase type 4, PDE I, PDEII, PDEIII, squalene cyclase inhibitor, CXCR1, CXCR2, nitric oxide (NO) synthase, cyclo-oxygenase 1, cyclo-oxygenase 2, 5HT receptors, dopamine receptors, G proteins ie Gq, histamine receptors, 5-lipoxygenase, tryptase serine protease, thymidylate synthase, purine nucleoside phosphorylase, GAPDH trypanosomal, glycogen phosphorylase, Carbonic anhydrase, chemokine receptors, JAK/STAT, RXR and similar, HIV 1 protease, HIV 1 integrase, influenza, neuraminidase, hepatitis B reverse transcriptase, sodium channel, multi drug resistance (MDR), protein P-glycoprotein (and MRP), tyrosine kinases, CD23, tyrosine kinase p56 1ck, CD4, CD5, IL-2 receptor, IL-1 receptor, TNF-alphaR, ICAM1, Ca++ channels, VCAM, VLA-4 integrin, selectins, CD40/CD40L, neurokinins and receptors, inosine monophosphate dehydrogenase, p38 MAP Kinase, Ras/Raf/MEK/ERK pathway, interleukin-1 converting enzyme, caspase, HCV, NS3 protease, HCV NS3 RNA helicase, glycinamide ribonucleotide formyl transferase, rhinovirus 3C protease, herpes simplex virus-1 (HSV-1), protease, cytomegalovirus (CMV) protease, poly (ADP-ribose) polymerase, cyclin dependent kinases, vascular endothelial growth factor, oxytocin receptor, microsomal transfer protein inhibitor, bile acid transport inhibitor, 5 alpha reductase inhibitors, angiotensin II, glycine receptor, noradrenaline reuptake receptor, endothelin receptors, neuropeptide Y and receptor, adenosine receptors, adenosine kinase and AMP deaminase, purinergic receptors (P2Y1, P2Y2, P2Y4, P2Y6, P2X1-7), farnesyltransferases, geranylgeranyl transferase, TrkA a receptor for NGF, beta-amyloid, tyrosine kinase Flk-1/KDR, vitronectin receptor, integrin receptor, Her-2/neu, telomerase inhibition, cytosolic phospholipase A2, EGF receptor tyrosine kinase.
Insecticide target examples include, ecdysone 20-monooxygenase, ion channel of the GABA gated chloride channel, acetylcholinesterase, voltage-sensitive sodium channel protein, calcium release channel, and chloride channels.
Herbicide target examples include Acetyl-CoA carboxylase, adenylosuccinate synthetase, protoporphyrinogen oxidase, and enolpyruvylshikimate-phosphate synthase.
These various targets are typically used in screens that look for a compound to alter the level of activity of the selected target and require the compound to be in solution. In some cases the assay to determine activity in a potential compound has to be based on a cell based assay. The best assays for compound screens are where the interaction of two molecules is modulated allowing the development of rapid assays based on the determination of binding.
In addition to the drawbacks of current drug and compound discovery efforts described above, problems of specificity arise due to the common basis for the activity of various compounds. For example in trying to find compounds which block the dopamine receptor, one is interested in the inhibition of a specific receptor sub-type due to its expression in a selected tissue. The binding site of the receptor is designed to bind to dopamine and thus has a common structure across the various sub-types of receptors. This homology of structure at the target site of the discovery effort makes it difficult to identify compounds with optimal levels of specificity for given sub-types and thus difficult to achieve the levels of therapeutic affect desired.
The present invention provides a solution to this problem.
Antigen Presentation
The target degradation of various proteins in the cell is a mechanism for the presentation of various peptides in the context of MHC. It has been demonstrated that the ubiquitination of intracellular proteins leads to the degradation of the protein via the 26S proteasome and enhanced presentation of the resultant peptides in the context of MHC I. This enhanced presentation leads to improved immune responses by the stimulation of various cells involved in the immune system. In many diseases the antigenicity of various proteins does not appear to be potent enough to generate a robust immune response. For example in the case of cancer certain antigens are present but fail to elicite a potent immune response (Tobery T and Siliciano R F., 1999, J Immunol. 162, 639-642). The present invention provides a solution to this problem of generating an improved immune response.
Antisense
Antisense technology is a novel drug therapy approach. Antisense drugs work at the genetic level to interrupt the process by which disease causing proteins are produced. Proteins play a central role in virtually every aspect of human metabolism. Many human diseases are the result of inappropriate protein production. Antisense drugs are designed to inhibit the production of disease causing proteins. These antisense drugs function by binding to specific nucleic acid sequences in a cell and block the production of specific proteins in this way a specific proteins level is reduced. Examples of targets for this technology are virus-based diseases, cancer, Crohn's disease, renal transplant rejection, psoriasis, ulcerative colitis, and inflammation. The specific targets are; HPV, HIV, CMV, hepatitis C, ICAM-1, PKC-alpha, c-raf kinase, Ha-ras, TNF-alpha and VLA-4.